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J. Biol. Chem., Vol. 281, Issue 26, 17977-17988, June 30, 2006

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Lys-D48 Is Required for Charge Stabilization, Rapid Flavin Reduction, and Internal Electron Transfer in the Catalytic Cycle of Dihydroorotate Dehydrogenase B of Lactococcus lactis^*

Jonathan P. Combe, Jaswir Basran, Parvinder Hothi, David Leys, Stephen E. J. Rigby^¶, Andrew W. Munro^||, and Nigel S. Scrutton¹

From the Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester M60 1QD, United Kingdom, the Department of Biochemistry, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester, LE1 7RH, United Kingdom, the ^¶Department of Biological Sciences, Queen Mary College, University of London, London E1 4NS, United Kingdom, and the ^||Manchester Interdisciplinary Biocentre, School of Chemical Engineering and Analytical Science, University of Manchester, Manchester M60 1QD, United Kingdom

Dihydroorotate dehydrogenase B (DHODB) catalyzes the oxidation of dihydroorotate (DHO) to orotate and is found in the pyrimidine biosynthetic pathway. The Lactococcus lactis enzyme is a dimer of heterodimers containing FMN, FAD, and a 2Fe-2S center. Lys-D48 is found in the catalytic subunit and its side-chain adopts different positions, influenced by ligand binding. Based on crystal structures of DHODB in the presence and absence of orotate, we hypothesized that Lys-D48 has a role in facilitating electron transfer in DHODB, specifically in stabilizing negative charge in the reduced FMN isoalloxazine ring. We show that mutagenesis of Lys-D48 to an alanine, arginine, glutamine, or glutamate residue (mutants K38A, K48R, K48Q, and K48E) impairs catalytic turnover substantially (50–500-fold reduction in turnover number). Stopped-flow studies demonstrate that loss of catalytic activity is attributed to poor rates of FMN reduction by substrate. Mutation also impairs electron transfer from the 2Fe-2S center to FMN. Addition of methylamine leads to partial rescue of flavin reduction activity. Nicotinamide coenzyme oxidation and reduction at the distal FAD site is unaffected by the mutations. Formation of the spin-interacting state between the FMN semiquinone-reduced 2Fe-2S centers observed in wild-type enzyme is retained in the mutant proteins, consistent with there being little perturbation of the superexchange paths that contribute to the efficiency of electron transfer between these cofactors. Our data suggest a key charge-stabilizing role for Lys-D48 during reduction of FMN by dihydroorotate, or by electron transfer from the 2Fe-2S center, and establish a common mechanism of FMN reduction in the single FMN-containing A-type and the complex multicenter B-type DHOD enzymes.

Received for publication, February 14, 2006 , and in revised form, April 3, 2006.

^* The work was funded by the UK Biotechnology and Biological Sciences Research Council and the Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplementary data.

¹ To whom correspondence should be addressed: Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, Jackson's Mill, PO Box 88, Sackville St., Manchester M60 1QD, United Kingdom. Tel.: 0161-306-5152; E-mail: nigel.scrutton{at}manchester.ac.uk.

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